Cannabis sativa L. (cannabis, hemp, marijuana) is one of the oldest and most versatile domesticated plants, which today finds use as source of medicinal, food, cosmetic and industrial products. It is also well known for its use as an illicit drug owing to its content of psychoactive cannabinoids (e.g. Δ9-tetrahydrocannabinol, Δ9-THC). Cannabinoids and other drugs that act through mammalian cannabinoid receptors are being explored for the treatment of diverse conditions such as chronic pain, multiple sclerosis and epilepsy.
Cannabinoids have their biosynthetic origins in both polyketide and terpenoid metabolism and are termed terpenophenolics or prenylated polyketides (Page J., Nagel J. (2006) Biosynthesis of terpenophenolics in hop and cannabis. In J T Romeo, ed, Integrative Plant Biochemistry, Vol. 40. Elsevier, Oxford, pp 179-210.). Cannabinoid biosynthesis occurs primarily in glandular trichomes that cover female flowers at a high density. Cannabinoids are formed by a three-step biosynthetic process: polyketide formation, aromatic prenylation and cyclization (see FIG. 1).
The first enzymatic step in cannabinoid biosynthesis is the formation of olivetolic acid by a polyketide synthase enzyme that catalyzes the condensation of hexanoyl-coenzyme A (CoA) with three molecules of malonyl-CoA. The major cannabinoids, including Δ9-tetrahydrocannabinolic acid and cannabidiolic acid, are formed from the precursor hexanoyl-CoA, which is a medium chain fatty acyl-CoA (see FIG. 1). Other cannabinoids with variant side-chains are formed from aliphatic-CoAs of different lengths (e.g. Δ9-tetrahydrocannabivarinic acid is formed from an n-butyryl-CoA primer).
Hexanoyl-CoA and other acyl-CoA thioesters in plants are synthesized by acyl-activating enzymes (AAEs, also called acyl-CoA synthetases) that catalyze the activation of carboxylic acid substrates using ATP. These enzymes act on a variety of carboxylate acids including short-, medium-, long- and very long-chain fatty acids, jasmonate precursors, phenylpropanoid-derived acids (e.g. cinnamic acid) and other organic acids such as malonate, acetate and citrate. Very few medium-chain acyl CoA synthetases have been previously identified in nature. In plants, three enzymes from A. thaliana, AAE7, At4g05160 and At5g63380 have been shown to form hexanoyl-CoA from hexanoate (Schneider K et al. (2005) A new type of peroxisomal acyl-coenzyme A synthetase from Arabidopsis thaliana has the catalytic capacity to activate biosynthetic precursors of jasmonic acid. The Journal of Biological Chemistry 280:13962-72; Shockey J M, Fulda M S, Browse J (2003) Arabidopsis contains a large superfamily of acyl-activating enzymes. Phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme a synthetases. Plant Physiology 132:1065-76.) Acyl-CoA synthetases from Pseudomonas spp. have been shown to act on medium-chain fatty acids such as hexanoate (Fernandez-Valverde M, Reglero A, Martinez-Blanco H, Luengo J M (1993) Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates. Applied Environmental Microbiology 59:1149-1154.)
Cannabinoids are valuable natural products. Genes encoding enzymes involved in cannabinoid biosynthesis will be useful in metabolic engineering of cannabis to produce plants that contain very low levels, or zero levels, of THCA and other cannabinoids via targeted mutagenesis (e.g. TILLING) or other gene knockout techniques. Such genes may also prove useful for creation, via marker-assisted selection, of specific cannabis varieties for the production of cannabinoid-based pharmaceuticals, or for reconstituting cannabinoid biosynthesis in heterologous organisms such as bacteria or yeast, or for producing cannabinoids in cell-free systems that utilize recombinant proteins.
Genes encoding enzymes of cannabinoid biosynthesis can also be useful in synthesis of cannabinoid analogs and synthesis of analogs of cannabinoid precursors. Cannabinoid analogs have been previously synthesized and may be useful as pharmaceutical products.
There remains a need in the art to identify enzymes, and nucleotide sequences encoding such enzymes, that are involved in the synthesis of aromatic polyketides.